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Isolated hepatocytes in culture represent an idoneous system for the study of liver physiology and metabolism. Furthermore, they are also widely utilized in pharmachological and toxychological study, in evaluating xenobiotic substance effects on the liver. In this paper, we reviewed the enzymatic methods for liver cell isolation in some mam malian species, as well as the techniques for qualitative and quantitative evaluation of cell number, vitality, purity, morphology and function. Recently, there has been a renewed interest in hepatocyte transplantation and hepatocyte-based liver support systems. From a clinical point of view, isolated hepatocytes could be useful in temporarily substituting an acutely damaged liver, a liver affected by a chronic pathology, or to correct an inherited liver disease carrying a severe metabolic derangement. Early experimental results of allogeneic hepatocyte transplantation, as well as the first clinical trials of bioartificial liver support systems employing xenogeneic hepatocytes are promising and contribute to maintain that interest in liver cell isolation and purification methods.